When MDCK cells (an epithelioid line derived from the kidney of a normal dog) are plated at high densities on a permeable and transluscent support, they develop an uninterrupted monolayer which has many of the structural and functional characteristics of a transporting epithelia (Cereijido et al, J. Cell Biol. 1978: 77, 853). The main two features of these cells are: 1) they contact each other and form tight junctions which confer to the monolayers the ability to act as effective permeability barriers; and 2) the apical and the basolateral aspects are structurally and functionally different, indicating that, as in natural transporting epithelia, cells are polarized. These characteristics are exhibited 15-20 hours after plating, and last up to a month. Our overall aim is to explore how these characteristics are installed and, the specific aim of this particular project is to explore: 1) the distribution and time course of formation of tight junctions (using a voltage scanning method); 2) the time course of the development of the Na/K selectivity of the apical border; 3) the time course of the installation of Na-pumping (by measuring Na-fluxes); 4) the time course of installation of the sensitivity to amiloride; 5) the role of the cytoskeleton in these processes (using fluorescent antibodies and inhibitors such as colchicine and cytochalasin-B); and 6) we will also test the effect of inhibitors of metabolism, and protein synthesis on both junction formation and polarization of the MDCK cells.